Composition for diagnosing parkinson&#39;s disease containing adipose tissue-derived mesenchymal stromal cell

ABSTRACT

The present invention relates to a composition for diagnosing a Parkinson&#39;s disease comprising mesenchymal stromal cells derived from adipose tissue, a method of providing information for diagnosing Parkinson&#39;s disease and/or the extent of the disease progression, a biomarker for diagnosing a Parkinson&#39;s disease, and a method of screening a drug candidate treating Parkinson&#39;s disease where the drug candidate is a target of the biomarker.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of Korean PatentApplication No. 10-2010-0058273 filed in the Korean IntellectualProperty Office on Jun. 18, 2010, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to a composition for diagnosing aParkinson's disease comprising mesenchymal stromal cells derived fromadipose tissue, a method of providing information for diagnosingParkinson's disease and/or extent of disease progression usingmesenchymal stromal cells derived from adipose tissue, a biomarker fordiagnosing a Parkinson's disease, and a method of screening an agenttreating Parkinson's disease where the agent targets the biomarker.

(b) Description of the Related Art

Parkinson's disease (PD) is a secondarily common neurodegenerativedisease which about one percent of old people aged over 60 suffers from,but the cause of disease has not yet been determined.

It has been suggested that Parkinson's disease has some connections withthe selective loss of dopaminergic neurons in substantia nigra, and withthe extensive neuron changes causing various complex motile and immotilesymptoms.

A genetic mutation of disease-causing gene such as α-synuclein, parkin,Parkinson disease autosomal recessive, early onset 7 (DJ-1), orphosphatase and tensin homologue (PTEN)-induced putative kinase 1(PINK1) is mentioned as a cause of familial Parkinson's disease.

Parkin acts as E3 ligase in ubiquitin-proteasomal system, protectsagainst the oxidative stress, and helps the maintenance of mitochondrialfunction. The mutation in Parkin gene can cause a hereditary early-onsetof Parkinson's disease.

The correlation between mitochondrial dysfunction and Parkinson'sdisease can be observed as the widely known disease-causing mechanism inPD patient subgroup involves aberrant shape and dysfunction ofmitochondria.

The damage of mitochondrial function increases an oxidative stress andassociates with the control of calcium homeostasis and cell apoptosispathway. The oxidative stress can be defined as one of causes inducingapoptosis of dopaminergic neuronal cells of the substantia nigra inParkinson's disease patient.

Parkinson's disease associated gene products including α-synuclein,Parkin, PINK1, DJ-1 and the like can be found in mitochondria and play acritical role in the mitochondrial dysfunction and oxidative stress.

The methods of diagnosing Parkinson's disease and determining extent ofthe disease progression include a method of imaging brain nigros-triatalregion with a magnetic resonance image (MRI) analysis, a positronemission tomography (PET), a single photon emission computed tomography(SPECT), and the like, and a method of analyzing a sample taken frombrain tissue with a biomarker. However, such methods still lead toinaccurate analysis of results, and unwanted pain and a risk to patientfrom directly taking a sample form brain tissue.

SUMMARY OF THE INVENTION

To solve the problems in the art, the present inventors found thatParkinson's disease could be diagnosed by using the mesenchymal stromalcell derived from adipose tissue, developed a technology for accuratelydiagnosing Parkinson's disease without directly taking brain tissue, andcompleted the present invention by developing a biomarker for diagnosingParkinson's disease using the technology.

Therefore, an embodiment of the present invention provides a compositionfor diagnosing Parkinson's disease comprising the mesenchymal stromalcell derived from adipose tissue.

Another embodiment provides a method of providing an information fordiagnosing Parkinson's disease and determining extent of the progressionof Parkinson's disease.

Further embodiment of the present invention provides a biomarker fordiagnosing Parkinson's disease where the biomarker is obtained from themesenchymal stromal cell derived from the adipose tissue.

Still another embodiment of the present invention provides a method ofscreening a drug treating Parkinson's disease where the drug targets thebiomarker.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention can provide a tool for understanding anddetermining the brain physiological states without isolating the braintissue from Parkinson's disease patient, by performing the transcriptomemicroarray analysis of mesenchymal stromal cell derived fromearly-passage adipose tissue taken from human subject with early-onset(congenital) hereditary Parkinson's disease such as Parkin-deficient PD,as well as late-onset (acquired) Idiopathic Parkinson's disease(idiopathic PD).

The present inventors completed the present invention by separating ahuman mesenchymal stromal cell derived from human adipose tissue(hAD-MSC), from a subject with Idiopathic PD or Parkin-deficientParkinson's disease, and comparing the gene expression pattern of themesenchymal stromal cell with that of non-Parkinson's disease. The humanadipose tissue is abundant and easily accessible source for mesenchymalstem cells (MSC).

Hereinafter, hAD-MSC obtained from a patient with idiopathic Parkinson'sdisease is referred to as ‘PD,’ hAD-MSC obtained from a patient withParkin-deficient Parkinson's disease as ‘Parkin,’ and hAD-MSC obtainedfrom a patient who has pituitary adenoma without Parkinson's disease as‘non-PD’ or ‘PA.’ Initially, by analyzing differentially-expressed gene(DEG) among three groups, 413 genes are confirmed to be differentiallyexpressed, and then classified into three groups of non-PD, PD andParkin. In addition, DEG are analyzed and divided to seven clustersaccording to K-mean clustering analysis, and the genbank accessionnumbers are listed in Tables 6a-6e. In addition, the functional groupsof human biomarker candidates are organized, and non-PD vs. PD andnon-PD vs. Parkin are compared. Finally, the PD associated DEGs whichare regulated differently due to the oxidative stress are classifiedinto one of groups among non-PD, PD and Parkin categories.

The knowledge of selective gene expression pattern in Parkinson'sdisease patient gained from the present invention can be helpful forobtaining the physiological information and early-diagnosis ofParkinson's disease, and developing an effective target specific drugfor treating the Parkinson's disease by using the genes as a biomarker.

First of all, the present invention relates to a composition fordiagnosing a disease, comprising mesenchymal stromal cells derived fromadipose tissue, where the disease is selected from the group consistingof Parkinson's disease, Alzheimer's disease, Huntington's disease,hereditary dystonia, hereditary dyskinesia, and metabolic disease. Thepresent inventors found that the presence of Parkinson's disease and theextent of disease progression could be diagnosed without separating thebrain tissue from Parkinson's disease patient by assaying the geneexpression pattern of mesenchymal stromal cell derived from adiposetissue, since the gene expression is very similar to that of braintissue. In accordance with the present invention, the use of adiposetissue-derived mesenchymal stromal cell for diagnosing the Parkinson'sdisease removes the step of dangerous separation of brain tissue fromthe subject. The diagnosing method using the adipose tissue-derivedmesenchymal stromal cells can be widely applied for diagnosing variousdiseases such as Alzheimer's disease, Huntington's disease, hereditarydystonia, hereditary dyskinesia, and metabolic disease.

The subject includes any kind of mammals, and preferably human beingswho are suffering or are likely to suffer from Parkinson's disease,Alzheimer's disease, Huntington's disease, hereditary dystonia,hereditary dyskinesia, and metabolic disease. Since one of the commoncauses of the diseases is adipose tissue (Human Lipodystrophies: Geneticand Acquired Diseases of Adipose Tissue andhttp://www.ncbi.nlm.nih.gov/pubmed/20551664), the application offollowing diagnosing technology for Parkinson's disease can be extendedto diseases listed above.

The adipose tissue can be one separated from mammals, preferably human.The separated region is not limited to a particular region of body andcan include any regions of a body such as breast and abdominal region.For more accurate analysis, the separated adipose tissue can be usedpreferably after eliminating cell debris and blood cell.

Hereinafter, the term, adipose tissue can be separated or unseparatedone from a live body, and includes an adipose cell.

In an embodiment of the present invention, the mesenchymal stromal cells(hAD-MSC) can be, for example, but are not limited thereto, cellsshowing the mononuclear cell properties which are confirmed by positiveexpression of human integrin beta-1 marker CD29, phagocyticglycoprotein-1 marker CD44, and human integrin alpha-4 marker CD49d, andat the same time expressing slightly primitive hematopoietic precursorsand vascular endothelial marker CD34, vascular endothelial marker CD31and vascular adhesion molecule 1, (VCAM-1) marker CD106.

The Parkinson's diseases which can be detected by using the compositionof the present invention includes all kinds of the Parkinson's diseasessuch as an acquired Idiopathic Parkinson's disease, congenital, familialParkinson's disease (for examples, Parkin, α-synuclein, phosphatase andtensin homologue (PTEN)-induced putative kinase 1 (PINK1, amitochondrial kinase), Parkinson disease autosomal recessive, earlyonset 7 (DJ-1), Leucine-rich repeat kinase 2 (LRRK2), and Hightemperature requirement protein A2 (HTRA2) deficient).

In another embodiment of the present invention, a method of diagnosingParkinson's disease using the mesenchymal stromal cells derived from theadipose tissue is provided.

More specifically, the method comprises the steps of separating amesenchymal stromal cell derived from adipose tissue of a subject,assaying a gene expression pattern of the mesenchymal stromal cell; anddetermining the presence of disease by analyzing the assayed result orthe disease progression degree by analyzing the assayed result.

The assaying step of gene expression pattern of hAD-MSC can be performedby any methods of analyzing gene expression which are used commonly inthe art. For examples, the assaying of gene expression can be carriedout by microarray analysis, Reverse transcriptase Polymerase ChainReaction (RT-PCR), Real time Polymerase Chain Reaction (real time PCR),genomics, proteomics, microRNA assay, SNP analysis, mitochondrial assay,functional assay and the like, but not limited thereto.

As a result of the molecular biological studies in the presentinvention, a high-throughput microarray analysis of hAD-MSC obtainedfrom a patient with Idiopathic Parkinson's disease, a patient withParkin-deficient Parkinson's disease, and a patient with non-Parkinson'sdisease can be established, and the analysis result is compared withthat of a patient with non-Parkinson's disease (control group) toidentify the gene groups which are differentially expressed between apatient with Idiopathic Parkinson's disease and/or a patient withParkin-deficient Parkinson's disease. Thus, the identified genes in apatient with non-Parkinson's disease, a patient with IdiopathicParkinson's disease and a patient with Parkin-deficient Parkinson'sdisease can contribute to the understanding of physiological symptoms ofParkinson's disease, and can provide a useful tool for developing theearly-diagnosis and effective treatment of Parkinson's disease targetingthe human biomarker.

The mitochondrial dysfunction and increased oxygen stress are shown insubgroup of Parkinson's disease patient, and these suggest the importanteffect of mitochondrial dysfunction and oxygen stress on onset ofParkinson's disease. Thus, the mitochondria can be effective target fordeveloping a biomarker of Parkinson's disease. The biochemical methodsof detecting the potential biomarker of Parkinson's disease include thegene screening method, mitochondria complex I measurement, blood levelof alpha-synuclein and isoforms measurement. Gene test tools which arecommercially available for detecting mitochondria mutant genes such asParkin, PINK1, and alpha-synuclein can be used. Furthermore, CoenzymeQ10, antioxidant and electron transporter (electron transporter chaincomponent) act as an electron transporter for mitochondria complex I andII.

The genes which show differential expression at least two times innon-PD patient vs. Parkin, and non-PD vs. PD, and Parkin vs. PD areidentified with hierarchical clustering analysis (FIG. 4), andsummarized in table 4 to select the PD-related genes (SCUBE3, IL8,ATP1B1, TNFRSF11B, FABP3, CXCL1). IL8 and CXCL1 are chemokines which acta basic role in development, homeostasis and immune systems, and involvein inflammation of cranial nerve of Parkinson's disease patient. SCUBE3accompanies an important molecule in dopaminergic neuron of ventralmidbrain, and TNFRSF11B involves in an inflammation in neurodegenerationof Parkinson's disease. The single heterozygous mutation of ATP1B1 issuggested to be related with a cause of early-onset of Parkinson'sdisease. Finally, FABP3 has been used as a diagnostic marker forParkinson's disease. The PD-related genes in non-PD vs. Parkin andParkin vs. PD are summarized in Table 5 (SYT14, LGR5, TGFB3, ITGA2,F2RL2, DRD1, PENK, GNA14, EDNRB, HSPA2, SLC6A6, AKR1B1, and PRG4). SYT14is a transmembrane protein involved with control of membranetrafficking. LGR5, F2RL2, DRD1, GNA14 and EDNRB involve in a signalpathway of G-protein, TGFB3 involves in a susceptibility of Parkinson'sdisease patient, and ITGA2 in a neuronal adhesion. An increasedexpression of PENK can be a cause of treatment-related dyskinesia inParkinson's disease patient. Parkin, as a substrate for parkin, mediatesthe ubiquitination of HSPA2 and a molecular chaperone. SLC6A6, alsoknown as taurine, is a neurotransmitter. Taurine is a beta-amino acidabundantly located in substantia nigra (SN), and functions as aneurotransmitter in substantia. The immunoreactivity of AKR1B1 isgenerated in human cerebral cortex and hippocampus, and PRG4 relateswith the inclusions of Parkinson's disease.

The comparison analysis of seven regulating sequences of K-meanclustering genes in non-PD, PD and Parkin are shown in FIG. 5 b. Thenames and genbank accession numbers of PD-related genes showing theincreased or decreased gene expression are summarized in Tables 6a-6e.The genes showing the increased gene expression pattern are shown inTable 6a (ITGA8, CTSH, CCRL1), Table 6b (TGFB3, DRD1, GNA14, PENK, PRG4,LGR5, HLA-DPA1), and Table 6e (SCUBE3, HSPA2, TGFB3, DRD1, GNA14, PENK,PRG4, LGR5, RELN, EDNRB, ITGA2, SLC6A6, F2RL2, CDK6, AKR1B1, MMP8, ID1,NEFM, ATP1B1, TNFRSF11B, TNFRSF10D), and the genes showing the decreasedgene expression pattern are shown in Table 6c (BEX1) and Table 6d (IL8,CXCL6). The change in gene expression seems to be due to themitochondria activity change between late-onset of IdiopathicParkinson's disease and congenital early-onset of Parkinson's disease ordue to compensation therebetween. These data also suggest the potentialbiomarker for the onset of Parkinson's disease. The molecular functionalgroups in non-PD vs. PD and non-PD vs. Parkin are determined and shownin FIG. 8 a to FIG. 8 d, and the genes which show the change in the geneexpression level in Idiopathic Parkinson's disease patient andParkin-deficient Parkinson's disease patient can provide a potentialhuman biomarker candidate for detecting a disease onset and a selectivevulnerability.

In addition, after analyzing of genes differentially expressed due tothe oxygen stress in PD, the genes and the clusters taken from ClusterNos. 2 to 6 are shown in Table 9. Interestingly, genes showing a linearincrease of gene expression were discovered in all groups, which may beexplained by an increased compensation against vulnerability caused byoxygen stress in pathology of Idiopathic PD and Parkin-deficient PD.

Based on these results, when there is an increased expression of atleast one selected from the group consisting of ITGA8, CTSH, CCRL1,TGFB3, DRD1, GNA14, PENK, PRG4, LGR5, HLA-DPA1, SCUBE3, HSPA2, RELN,EDNRB, ITGA2, SLC6A6, F2RL2, CDK6, AKR1B1, MMP8, ID1, NEFM, ATP1B1,TNFRSF11B, and TNFRSF10D in mesenchymal stromal cells derived fromadipose cell of a patent, or when where is an decreased expression of atleast one selected from the group consisting of BEX1, IL8, and CXCL6 inmesenchymal stromal cells derived from adipose cell of a patent, it ispossible to determine the presence of the Parkinson's Disease.Parkinson's disease includes late-onset (acquired) Parkinson's disease(Idiopathic Parkinson's disease), or early-onset (congenital, familial,hereditary) Parkinson's disease (for examples, Parkin, α-synuclein,phosphatase and tensin homologue (PTEN)-induced putative kinase 1(PINK1, a mitochondrial kinase), Parkinson disease autosomal recessive,early onset 7 (DJ-1), Leucine-rich repeat kinase 2 (LRRK2), or Hightemperature requirement protein A2 (HTRA2) deficient Parkinson's diseaseand etc).

In an embodiment of the present invention, the increase and decrease ofthe gene expressions can be measured by the amount of protein expressed.When the amount is about 1.5 to 3 times higher than that of normal groupwithout Parkinson's disease, the result can be determined to besignificant.

In an embodiment of the present invention, a method of screening a drugtreating Parkinson's disease, in which the drug targets the biomarker inmesenchymal stromal cells derived from adipose tissue, is provided.

More specifically, the method comprises the steps of contacting a drugcandidate with a mesenchymal stromal cell derived from adipose tissue;and assaying a gene expression pattern of the mesenchymal stromal cell;and determining the drug candidate as a drug treating Parkinson'sdisease in case that there is a difference of gene expression pattern inthe mesenchymal stromal cells between the treatment and non-treatment ofthe drug candidate, wherein at least gene is selected from the groupconsisting of ITGA8, CTSH, CCRL1, TGFB3, DRD1, GNA14, PENK, PRG4, LGR5,HLA-DPA1, SCUBE3, HSPA2, RELN, EDNRB, ITGA2, SLC6A6, F2RL2, CDK6,AKR1B1, MMP8, ID1, NEFM, ATP1B1, TNFRSF11B, TNFRSF10D, BEX1, IL8, andCXCL6.

For example, when the group treated with the drug candidate shows theincreased gene expression (preferably, at least an increase of two-fold)of at least one selected from the group consisting of ITGA8, CTSH,CCRL1, TGFB3, DRD1, GNA14, PENK, PRG4, LGR5, HLA-DPA1, SCUBE3, HSPA2,RELN, EDNRB, ITGA2, SLC6A6, F2RL2, CDK6, AKR1B1, MMP8, ID1, NEFM,ATP1B1, TNFRSF11B, TNFRSF10D and the like, or the decreased geneexpression (preferably, at least a decrease of two-fold) of at least oneselected from the group consisting of BEX1, IL8, CXCL6 and the like,compared to that of the group untreated with the drug candidate, thedrug candidate can be determined as a drug for treating Parkinson'sdisease, for examples, late-onset (acquired) Parkinson's Disease(Idiopathic Parkinson's Disease), or early-onset (congenital, familial,hereditary) Parkinson's Disease (for examples, Parkin, α-synuclein,phosphatase and tensin homologue (PTEN)-induced putative kinase 1(PINK1, a mitochondrial kinase), Parkinson disease autosomal recessive,early onset 7 (DJ-1), Leucine-rich repeat kinase 2 (LRRK2), or Hightemperature requirement protein A2 (HTRA2) deficient Parkinson'sdisease, etc.

The increase or decrease of gene expression can be measured by anymethod of measuring the gene expression level which has been knowngenerally in the art, for examples but not limited to, microarray assay,Reverse transcriptase polymerase chain reaction (RT-PCR), Real time PCR,genomics, proteomics, microRNA assay, SNP analysis, mitochondrial assay,functional assay and the like.

The data obtained in the present invention provide a predictablescenario for onset of Parkinson's disease. In conclusion, the geneexpression analysis of the mesenchymal stromal cells derived from thehuman adipose tissue can identify specific molecular functional groupsof the genes which are affected by the mitochondrial dysfunction andoxidative stress. Thus, the present invention provides a technology fordiagnosing Parkinson's disease and/or determining its extent of diseaseprogression using the mesenchymal stromal cell derived from adiposetissue instead of brain tissue, for. The genes which show the change ingene expression in Idiopathic or Parkin-deficient (familial) Parkinson'sdisease patient compared to the non-Parkinson's disease can beidentified by using such technology, and can be used both as a biomarkeras well as a target for developing a drug treating Parkinson's disease.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 a and 1 b show the procedure of separating and culturing themesenchymal stromal cells derived from adipose tissue of human patientwith Parkinson's disease.

FIG. 2 represents schematically the procedure of preparing a stock byculturing the mesenchymal stromal cells derived from adipose tissue ofhuman patient with Parkinson's disease.

FIG. 3 is a picture showing the transient change in cell morphology ofthe mesenchymal stromal cells derived from adipose tissue of humanpatient with Parkinson's disease based on culture time variations.

FIG. 4 is a Venn diagram of Differentially Expressed Gene (DEG) betweenthe control group (non-PD, PA) and test group with Parkinson's disease.

FIG. 5 a is a result of hierarchical clustering analysis of the genesshowing the gene expression level at least two times higher betweennon-PD, PD and Parkin groups.

FIG. 5 b shows the seven clusters obtained by reorganizing theexpression pattern graphs which are classified through the K-meanclustering analysis of non-PD, PD and Parkin.

FIG. 6 represents the clustering analysis result of the genes showinglinearly-decreased gene expression between non-PD, PD, and Parkingroups.

FIG. 7 represents the clustering analysis result of the genes showinglinearly-increased gene expression between non-PD, PD, and Parkingroups.

FIG. 8 a to 8 d show the result classifying human biomarker which areobtained by reprogramming the molecular functional groups by using GeneOntology and Panther database system between non-PD vs. PD and non-PDvs. Parkin.

FIG. 9 shows the comparison of cell morphology before and afterimmortalization with hTERT in non-PD, PD, and Parkin group.

After: the cell morphology shortly after immortalization with hTERT.

6 months: the cell morphology after immortalization with hTERT andculturing for 6 months.

1 year: the cell morphology after immortalization with hTERT andculturing for 1 year.

FIG. 10 shows a result of chromosomal structure (karyotype) analysis.

after: the karyotype analysis result shortly after immortalization withhTERT.

1 year: the karyotype analysis result after immortalization with hTERTand culturing for 1 year.

FIG. 11 a shows a result of biochemical enzyme assay of mitochondriarespiration chain of immortalized cell, and FIG. 11 b is the result ofwestern blot analysis showing successful separation of mitochondria inthe assay of FIG. 11 a.

FIG. 12 is electronic microscopic images of mitochondria inearly-culture stage and immortalization-culture stage of the cells (a,b: mitochondria in Non-PD; c, d: mitochondria in PD; and e, f:mitochondria in Parkin).

FIG. 13 a represents a result of western blot analysis (left side) whichshows the change in the gene expression of mitochondrial markers inearly-culture stage of non-PD, PD, and Parkin groups, and a graph (rightside) showing the quantitative analysis of the result.

FIG. 13 b represents the change in the gene expression of HSP60 asmitochondria marker in immortalization-culture stage of the cells innon-PD, PD, and Parkin (left side) and a graph (right side) showing thequantitative analysis of the result.

FIG. 13 c represents the change in the gene expression of HSP90 asmitochondria marker in immortalization-culture stage of the cells innon-PD, PD, and Parkin (left side) and a graph (right side) showing thequantitative analysis of the result.

FIG. 14 a represents a western blot analysis showing the change in thegene expression of autophagy makers in immortalization-culture stage ofthe cells in non-PD, PD, and Parkin.

FIG. 14 b represents a western blot analysis showing the change in thegene expression of mTOR as an autophagy maker in immortalization-culturestage of the cells in non-PD, PD, and Parkin of FIG. 14 a.

FIG. 14 c represents a western blot analysis showing the change in thegene expression of S6K as an autophagy maker in immortalization-culturestage of the cells in non-PD, PD, and Parkin of FIG. 14 a.

FIG. 15 a represents a western blot analysis showing the change in thegene expression of autophagy markers in immortalization-culture stage ofthe cells in non-PD, PD, and Parkin, and FIG. 15 b is a graph showingthe quantitative analysis of the result.

FIG. 16 a represents western blot analysis showing the change in thegene expression of autophagy markers in immortalization-culture stage ofthe cells in non-PD, PD, and Parkin.

FIG. 16 b is a graph showing the quantitative analysis of the change inthe gene expression of autophagy markers in immortalization-culturestage of the cells in non-PD, PD, and Parkin.

FIG. 17 shows an analysis result of properties of the mesenchymalstromal cells derived from the adipose tissue.

EXAMPLE

The present invention is further explained in more detail with referenceto the following examples. These examples, however, should not beinterpreted as limiting the scope of the present invention in anymanner.

Example 1 Preparation of the Mesenchymal Stromal Cell Derived from theAdipose Tissue

1.1. Separation and Culture of the Mesenchymal Stromal Cells Derivedfrom the Adipose Tissue

The adipose tissue-derived mesenchymal stromal cells (hAD-MSC) wereseparated from Idiopathic Parkinson's disease (Idiopathic PD) patient,Parkin-deficient Parkinson's disease (Parkin-deficient PD) patient, anda pituitary adenoma patient who did not suffer from Parkinson's disease,and then subsequently were cultured. Hereinafter, otherwise particularlydefined, hAD-MSC obtained from Idiopathic PD patient is referred to as“PD,” hAD-MSC obtained from Parkin-deficient Parkinson's disease patientto as “Parkin”, and hAD-MSC obtained from Parkinson's disease to as“non-PD” or “PA.”

These tests were performed under the permission of Institutional ReviewBoard of Seoul National University Hospital (IRB No. 0707-024-212) andthe written consent of the patients. During the performance of DeepBrain Stimulation (DBS) surgery of Idiopathic PD patient, andParkin-deficient PD patient, the adipose tissue under the skin ofclavicle was taken in order to compare with that of a pituitary adenomapatient (control group) who did not suffer from Parkinson's disease.

The adipose tissue was added to 1% antibiotic/antimyotic(Gibco®Invitrogen, Carlsbad, Calif.) in sterilized PBS(phosphate-buffered saline, pH7.4) and transferred to test room. Theadipose tissue was washed with PBS three times to remove tissue debrisand red blood cell, and then finely cut into small pieces. The adiposetissue was digested with 0.075% collagenase Type I (Sigma-Aldrich, St.Louis, Mo., USA) at 37° C. for 1 hour, inactivated with the same volumeof DMEM/10% Fetal Bovine Serum (FBS) (Gibco® Invitrogen, Carlsbad,Calif.), and centrifuged at 1200×g for 10 minutes. The obtained pelletwas cultured in three times volume of red blood lysis buffer (QIAGEN,valencia, CA, USA) at 37° C. for 10 minutes, and filtered with 100 μMstrainer. The filtrate was centrifuged at 1200×g for 10 minutes. Theresultant pellet was washed with PBS and centrifuged at 1200×g for 10minutes. Finally, the obtained pellet was resuspended in MesenchymalStem cell Expansion medium (Millipore, SCM015, Billerica, Mass., USA)and placed onto 25T culture flask. After the cells were cultured inMesenchymal Stem cell Expansion medium (Millipore, SCM015, Billerica,Mass., USA) at 37° C. for 48 hours, the cells were washed with PBS andthe unattached cells were removed. The culture medium was replaced withnew medium every three days.

As a result, the hAD-MSC was obtained. Among the hAD-MSC obtained, twocells obtained from a pituitary adenoma patient who did not suffer fromParkinson's Disease (PA1 and PA2), two cells obtained from Idiopathic PD(PD1 and PD2), and two cells obtained from Parkin-deficient PD (Parkin)and Parking) were deposited at Korean cell line bank located at 28Yongon-dong, Chongno-gu, Seoul 110-744, Korea on Nov. 17, 2010, and thenassigned with the accession numbers of KCLRF-BP-00239(PA1),KCLRF-BP-00240(PA2), KCLRF-BP-00241(PD1), KCLRF-BP-00242(PD2),KCLRF-BP-00243(Pakin1), and KCLRF-BP-00244(Pakin2).

The culture procedure is shown schematically in FIGS. 1 a and 1 b. FIG.2 represents schematically the procedure of preparing stock by culturingmesenchymal stromal cells derived from adipose tissue of human patientwith Parkinson's disease. FIG. 3 is a picture showing the transientchange in cell morphology of mesenchymal stromal cells derived fromadipose tissue of human patient with Parkinson's disease according tothe culture time variations. In FIG. 3, the pictures of cell morphologyof adipose tissue-derived mesenchymal stromal cells were taken every dayduring continuous culture.

The information on the cell culture of mesenchymal stromal cells derivedfrom adipose tissue obtained from Idiopathic PD patient,Parkin-deficient PD patient, and control group are summarized at Tables1 to 3:

TABLE 1 Culture information of adipose tissue obtained from IdiopathicPD patient Patient No. Labeling Culture Date 1 FSC-PD#2 2007-03-09 2FSC-PD#3 2007-04-02 3 FSC-PD#5 2007-08-13 4 FSC-PD#6 2007-10-22 5FSC-PD#7 2007-10-29 6 FSC-PD#8 2007-11-19 7 FSC-PD#9 2008-03-24 8FSC-PD#10 2008-07-07 9 FSC-PD#11 2008-08-29 10 FSC0714 2008-07-14 11FSC0721 2008-07-21 12 FSC0829 2008-08-29 13 FSC1006 2008-10-06 14FSC0119 2009-01-19 15 FSC0209 2009-02-09 16 FSC0420 2009-04-20 17FSC0427 2009-04-27 18 FSC0601 2009-06-01 19 FSC0622 2009-06-22 20FSC0918 2009-09-18 21 FSC1123 2009-11-23

TABLE 2 Culture information of adipose tissue obtained fromParkin-deficient PD patient Patient Labeling Culture Date 1 FSC-parkin2007-05-17 2 gFSC 2008-06-02

TABLE 3 Culture information of adipose tissue obtained from controlgroup Patient No. Labeling Culture Date 1 FSC-#1 2006-11-22 2 FSC-#22006-11-23 3 FSC-#3 2006-12-18 4 FSC-#4 2006-12-18 5 FSC-#7 2007-01-08 6FSC-#8 2007-01-22 7 FSC-#9 2007-01-31 8 FSC-#11 2007-02-15 9 FSC-#122007-02-26 10 FSC-#14 2007-03-15 11 FSC-#15 2007-04-20 12 FSC-#172007-10-02 13 FSC-#18 2007-10-04 14 FSC-#19 2008-03-25 15 FSC10132008-10-13 16 FSC1014 2008-10-14 17 FSC1103 2008-11-03 18 FSC11042008-11-04 19 FSC0629 2009-06-29 20 FSC0630 2009-06-30 21 FSC07062009-07-06 22 FSC1019 2009-10-19 23 FSC1102 2009-11-02 24 FSC11092009-11-09 25 FSC1201 2009-12-01

1.2. Fluorescence-Activated Cell Sorter (FACS) Analysis

The hAD-MSC culture was separated with PBS and subsequently culturedwith following primary antibodies (culture medium: Mesenchymal Stem cellExpansion medium (Millipore, SCM015, Billerica, Mass., USA), culturetemperature: 37° C.).

Primary Antibody:

anti-CD29, anti-CD44, anti-CD34, anti-CD31 (DakoCytomation, Carpinteria,Calif., USA), anti- and CD49d, anti-CD106 (Chemicon, Temecula, Calif.,USA).

The cells were cultured on ice for 30 minutes, and then washed with 0.5%BSA and 2 mM EDTA in BSA (Sigma-Aldrich, St. Louis, Mo., USA). Themorphological characteristics of hAD-MSC and quantitative analysis wereperformed by FACS SCAN flow cytometer (Becton Dickinson, San Diego,Calif., USA) and CellQuest software (Becton Dickinson, San Diego,Calif., USA).

The results are shown in FIG. 17. hAD-MSC obtained from idiopathic PDpatient, Parkin-deficient PD patient, and control group were separatedand cultured. Then, the cells showed the characteristics of mononuclearcell based on the expression of human integrin beta-1 marker CD29,phagocytic glycoprotein-1 marker CD44, and human integrin alpha-4 markerCD49d. In addition, the cells expressed slightly primitive hematopoieticprecursors and vascular endothelial marker CD34, vascular endothelialmarker CD31 and vascular adhesion molecule 1, (VCAM-1) marker CD106.

1.3. Preparation of RNA Sample

According to manufacturer's manual, the RNA sample was prepared.Specifically, whole RNA was separated with RNeasy Mini Kit columns(Qiagen, Hilden, Germany) according to the manufacturer's manual. Thequantity of RNA was assessed with Agilent 2100 bioanalyser using RNA6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands) anddetermined with ND-1000 Spectrophotometer (Nanoprop Technologies, Inc.,DE, USA).

1.4. Analysis of hAD-MSC Properties

hAD-MSC was separated from Idiopathic PD patient, Parkin-deficient PDpatient, and the control group and cultured. In flow cytometry analysis,at least 95% of MSC (≧95%) expressed CD105, CD73 and CD90, and the cellswere deficient in the expression of CD45, CD34, CD14 or CD11b; CD79□ orCD19; and HLA class II (positive at most 2%) (M Dominici, K Le Blanc, IMueller, I Slaper-Cortenbach, F Marini, D Krause, et al, Minimalcriteria for defining multipotent mesenchymal stromal cells. TheInternational Society for Cellular Therapy position statement,Cytotherapy, 8, 315-7, 2006). The accession numbers of the genes aresummarized in following table:

Gene name Gene accession number CD105 NM_001114753 CD73 NM_002526 CD90NM_006288 NM_033209 CD45 NM_002838 CD34 NM_001025109 CD14 NM_000591CD11b NM_000632 CD79 NM_001783 CD19 NM_001178098 HLA class II NM_000449

The cells should be able to differentiate into osteoblasts cell, adiposecell (adipocytes) and chondroblasts under the standard in vitrodifferentiation condition (M Dominici, K Le Blanc, I Mueller, ISlaper-Cortenbach, F Marini, D Krause, et al, Minimal criteria fordefining multipotent mesenchymal stromal cells. The InternationalSociety for Cellular Therapy position statement, Cytotherapy, 8, 315-7,2006).

The separated hAD-MSC were cultured at Mesenchymal Stem cell Expansionmedium (Millipore, SCM015, Billerica, Mass., USA) at 37° C., and theexpression profile of human integrin beta-1 marker CD29, phagocyticglycoprotein-1 marker CD44, and human integrin alpha-4 marker CD49d wereanalyzed with FACS analysis method. The result is shown in FIG. 17. CONin FIG. 17 shows FACS analysis result of mesenchymal stromal cellsthemselves.

The accession numbers of the genes utilized are summarized in followingtable:

Gene name Gene accession number CD29 NM_002211 CD44 NM_000610 CD49dNM_000885 CD34 NM_001025109 CD31 NM_000558 CD106 NM_001078

The positive expression of the genes confirmed that the cells showed theproperties of the mononuclear cell. The cells expressed a small amountof primitive hematopoietic precursors and vascular endothelial markerCD34, vascular endothelial marker CD31 and vascular adhesion molecule 1,(VCAM-1) marker CD106.

Example 2 Gene Profiling of Adipose Tissue-Derived Mesenchymal StromalCell

2.1. cDNA Microarray Analysis

The gene expression analysis was performed with Affymetrix GeneChip®Human Gene 1.0 ST oligonucleotide array (DNA LINK, INC (Seoul, Korea)).

According to Affymetrix manufactor's protocol(http://www.affymetrix.com), 300 ng of RNA was added to each sample.Namely, 300 ng of all RNA per a sample was changed into double strandedcDNA. The double stranded cDNA was obtained by using SuperScrpit IIReverse Transcriptase, DNA polymerase I and random hexamer inserted byT7 promoter (Affymetrix GeneChip® WT cDNA Synthesis and AmplipicaitionKit, Cat No. 900672). An amplified RNA (cDNA) was produced by in vitrotranscription (IVT) with IVT Enzyme Mix (Affymetrix GeneChip® WT cDNASynthesis and Amplipicaition Kit, Cat No. 900672), and separated withAffymetrix sample cleanup module. The amplified cRNA was mixed with IVTcRNA binding buffer and 100% EtOH, and bonded in cRNA cleanup spincolumn. The column was washed with cRNA wash buffer, and then elutedwith RNase-free water.

cDNA was reproduced with dNTP mixture including dUTP AffymetrixGeneChip® WT cDNA Synthesis and Amplipicaition Kit, Cat No. 900672),according to the random-primed reverse transcription. Then, the producedcDNA was fragmented by using UDG and APE 1 restriction enzyme(Affymetrix GeneChip® WT Terminal Labeling Kit, Cat No. 900670), and theend labeling was performed by inserting biotinylated dideoxynucleotidewith TdT (Terminal deoxynucleotidyl transferase) enzyme.

According to Gene Chip Whole Transcript (WT) Sense Target Labeling AssayManual (Affymetrix), the end-labeled and fragmented cDNA was hybridizedat 60 r/min, at 45° C., for 16 hours with GeneChip® Human Gene 1.0 STarray. Then, the array was stained and washed in Genechip FluidicsStation 450 (Affymetrix), and scanned with Genechip Array scanner 30007G (Affymetrix).

2.2. Classification of DEG Included in Three Groups of Non-PD (ControlGroup), Idiopathic PD and Parkin-Deficient PD

To identify DEG based on two-fold gene expression difference betweennon-PD vs. Parkin, non-PD vs. PD and Parkin vs. PD, hierarchicalclustering analysis (Eisen M B, Spellman P T, Brown P O, Botstein D,1998) Cluster analysis and display of genome-wide expression patterns.Genetics Vol. 95, Issue 25, 14863-14868) was performed. The hierarchicalclustering analysis is data-mining algorithm used for definingsimilarity or dissimilarity of expressed genes. By using all genesshowing the gene expression difference of two-fold between three groupsof non-PD, PD, Parkin as a standard, the hierarchical clusteringanalysis was carried out to identify the gene having high similarity andthe Euclidean distance was used as a similarity measurement.

The obtain result is shown as a Venn diagram in FIG. 4. In order toidentify genes showing expression level difference of at least two-foldbetween the groups of non-PD, PD and Parkin, after selecting genesshowing expression level difference of at least two-fold in each non-PDvs Parkin, non-PD vs PD, and Parkin vs PD, and comparing with theresults among non-PD vs Parkin, non-PD vs PD, and Parkin vs PD, thegenes that showed the similar level of difference in each comparisonwere selected and shown in FIG. 4.

For example, the genes having gene expression level of at least twotimes higher between the groups of non-PD vs. Parkin were 109 geneswhich included 20 genes showing two-fold expression in comparison ofnon-PD vs. Parkin and 16 genes showing two-fold expression level in allthree comparisons.

Differentially-expressed genes were 413 genes where 109 genes were fornon-PD vs. Parkin, 233 genes for non-PD vs. PD and 335 genes for Parkinvs. PD. Particularly, 6 genes which had been already known as PD-relatedgene were selected from 16 common genes in the center of non-PD vs.Parkin vs. PD, and their genbank accession numbers are listed in Table4.

TABLE 4 Fold Change (log2 ratio) Genbank non-PD non-PD Parkin Accessionvs. vs. vs. No. Gene name (Gene symbol) Parkin PD PD Function NM_152753signal peptide, CUB domain, EGF-like 3 2.0097 −1.6452 −3.6549 proteinhetero- (SCUBE3) homo-oligomerization NM_000584 interleukin 8 (IL8)−1.5026 −3.5819 −2.0792 angiogenesis/ cell motility NM_001677 ATPase,Na⁺/K⁺ transporting, β1 1.1420 −2.1375 −3.2796 ion transport polypeptide(ATP1B1) NM_002546 tumor necrosis factor receptor 1.2471 −1.6420 −2.8891apoptosis/ superfamily, member 11b (TNFRSF11B) inflammation responseNM_004102 fatty acid binding protein 3, muscle and −1.4108 1.7890 3.1998phosphatidylcholine heart (mammary-derived growth biosynthetic processinhibitor) (FABP3) NM_001511 chemokine (C—X—C motif) ligand 1□ −1.0412−2.9751 −1.9339 chemotaxis/ (melanoma growth stimulating activity,immune response α) (CXCL1)

In addition, 13 genes which have been known as Parkinson'sdisease-related gene are selected from 56 genes in groups of non-PD vs.Parkin and Parkin vs. PD, and their genbank accession numbers are shownin Table 5. The genes do not include 16 genes which are common in threegroups.

TABLE 5 Fold Change Genbank (log2 ratio) Accession non-PD Parkin No Genename (Gene symbol) vs. Parkin vs. PD Function NM_153262 synaptotagminXIV (SYT14) 1.6903 −1.5708 membrane trafficking NM_003667 leucine-richrepeat-containing G protein- 2.1567 −2.1239 G-protein signaling coupledreceptor 5 (LGR5) NM_003239 transforming growth factor, β3 (TGFB3)1.1171 −1.0345 cell growth/aging NM_002203 integrin α2 (ITGA2) 1.3831−1.8448 cell adhesion NM_004101 coagulation factor II (thrombin)receptor- 1.6712 −2.2182 G-protein signaling like 2 (F2RL2) NM_000794dopamine receptor D1 (DRD1) 1.1496 −1.1496 G-protein signaling NM_006211proenkephalin (PENK) 1.9852 −1.9852 neuropeptide signaling NM_004297 Gprotein α14 (GNA14) 2.5842 −2.5842 G-protein signaling NM_001122659endothelin receptor type B (EDNRB) 1.0755 −1.6347 G-protein signalingNM_021979 heat shock 70 kDa protein 2 (HSPA2) 1.0932 −1.4480 response tounfolded protein NM_003043 solute carrier family 6, member 6 1.0595−1.3053 amino acid (SLC6A6) metabolic process NM_001628 aldo-ketoreductase family 1, member B1 1.1052 −1.3105 metabolic process (aldosereductase) (AKR1B1) NM_005807 proteoglycan 4 (PRG4) 3.8210 −4.4772 cellproliferation

2.3. Clustering Analysis and Result

After finally washing and staining, the image was scanned withAffymetrix GeneChip® Human Gene 1.0 ST array using Affymetrix Model 3000G7 scanner. The image data was extracted by Affymetrix Commnad Consolesoftware1.1. The raw excel file was used for obtaining the expressionextent data in the next step. The expression data was obtained byExpression Console software version 1.1 (www.affymetrix.com). The datanormalization was performed with Robust Multi-Average (RMA) algorithm inExpression Console software. The genes showing the increase of geneexpression level of at least two fold between the test group and thecontrol group were selected and used in the subsequent step.

The gene expression level of the selected genes was measured withHierarchical clustering in MEV (MultiExperiment Viewer) software4.0(http://www.tm4.org, TM4: a free, open-source system for microarraydata management and analysis. Biotechniques. 2003 February;34(2):374-8.). To classify the genes as common gene groups havingsimilar expression pattern, K-mean Clustering (http://www.tm4.org) wasperformed (A Soukas, P Cohen, N D Socci, J M Friedman, Leptin-specificpatterns of gene expression in white adipose tissue, Genes Dev, 14,963-80 (2000)). The K-mean Clustering is a method used for classifying,based on their patterns, the common gene expression groups having thegenes of similar expression pattern in hierarchical clustering analysis.

The genes which are differentially expressed were analyzed biologicallywith Web-based DAVID (the Database for Annotation, Visualization, andIntegrated Discovery; http://david.abcc.ncifcrf.gov/home.jsp, Systematicand integrative analysis of large gene lists using DAVID BioinformaticsResources. (2009) Nat. Protoc. 4(1):44-57.). The genes were classifiedon the basis of Gene ontology, Panther ontology database(http://david.abcc.ncifcrf.gov/home.jsp).

Based on the similarity measured by the K-mean clustering analysisbetween non-PD, PD and Parkin groups, the expressed genes wereclassified. The expression pattern graphs were reorganized to sevenclusters (FIG. 5 b).

FIG. 5 a showed a result of Hierarchical Clustering by using the signalsof 413 genes which represent the difference in gene expression betweenthe groups of PA, PD, and Parkin. The result confirmed the whole profileof clusters showing the difference in gene expression between the groupsof PA, PD, and Parkin, and the expected several patterns of theclusters. FIG. 5 b showed classified clusters showing similar geneexpression pattern, when 7 patterns of clusters were classifiedaccording to the result of hierarchical clustering analysis. The numberof pattern was determined by the smallest optimized number which wasobtained after performing repetitive simulation with various patternnumbers.

Specifically, the gene names and their genbank accession number ofCluster 2, 3, 4, 5 and 6 were summarized in Tables 6a-6e.

TABLE 6a Cluster 2: Increase Genbank no Gene name (non-PD < PD = Parkin)Gene symbol accession No 1 interleukin α8 ITGA8 NM_003638 2 cathepsin HCTSH NM_004390 3 chemokine (C-C motif) receptor-like 1 CCRL1 NM_178445

TABLE 6b Cluster 3: Increase Genbank no Gene name (non-PD ≦ PD < Parkin)Gene symbol accession No 1 transforming growth factor, β3 TGFB3NM_003239 2 dopamine receptor D1 DRD1 NM_000794 3 G protein α14 GNA14NM_004297 4 proenkephalin PENK NM_006211 5 proteoglycan 4 PRG4 NM_0058076 leucine-rich repeat-containing LGR5 NM_003667 G-protein coupledreceptor 5 7 major histocompatibility HLA-DPA1 NM_033554 complex, classII, DP α1

TABLE 6c Cluster 4: Decrease Genbank no Gene name (non-PD ≧ PD > Parkin)Gene symbol accession No 1 brain expressed, X-linked 1 BEX1 NM_018476

TABLE 6d Cluster 5: Decrease Genbank no Gene name (non-PD > PD = Parkin)Gene symbol accession No 1 interleukin 8 IL8 NM_000584 2 chemokine(C—X—C motif) ligand 6 CXCL6 NM_002993 (granulocyte chemotactic protein2)

TABLE 6e Cluster 6: Increase Gene Genbank no Gene name (non-PD ≧ PD <Parkin) symbol accession No 1 signal peptide, CUB domain, EGF-like 3SCUBE3 NM_152753 2 heat shock 70 kDa protein 2 HSPA2 NM_021979 3transforming growth factor, β3 TGFB3 NM_003239 4 dopamine receptor D1DRD1 NM_000794 5 G protein α14 GNA14 NM_004297 6 proenkephalin PENKNM_006211 7 proteoglycan 4 PRG4 NM_005807 8 leucine-richrepeat-containing G-protein coupled receptor 5 LGR5 NM_003667 9 reelinRELN NM_005045 10 endothelin receptor type B EDNRB NM_001122659 11Integrin α2 ITGA2 NM_002203 12 solute carrier family 6, member 6 SLC6A6NM_003043 13 coagulation factor II (thrombin) receptor-like 2 F2RL2NM_004101 14 cyclin-dependent kinase 6 CDK6 NM_001259 15 aldo-ketoreductase family 1, member B1 (aldose reductase) AKR1B1 NM_001628 16matrix metallopeptidease 8 MMP8 NM_002424 17 inhibitor of DNA binding 1,dominant negative helix-loop- ID1 NM_181353 helix protein 18neurofilament, medium polypeptide NEFM NM_005382 19 ATPase, Na⁺/K⁺transporting, β1 polypeptide ATP1B1 NM_001677 20 tumor necrosis factorreceptor superfamily, member 11b TNFRSF11B NM_002546 21 tumor necrosisfactor receptor superfamily, member 10d, TNFRSF10D NM_003840 decoy withtruncated death domain

The increased pattern of gene expression was shown in Clusters 2, 3, and6, and the decreased pattern was shown in Clusters 4 and 5. The genedata showing greatest linear-increase of gene expression (Cluster 3) andthe genes showing greatest linear-decrease of gene expression (Cluster4) could provide the numerical values of severe Parkinson's disease anda guidance for a search for the human biomarker diagnosing early-stageof Parkinson's disease.

The gene expression result is described in detail hereinafter.

Firstly, the genes which showed the linear decrease of gene expressionamount in the order of PA>PD>Parkin were Cluster 4 in Table 7 and FIG.6.

TABLE 7 Gene name Function Genbank accession # brain expressed,multicellular organismal NM_018476 X-linked 1 development // nervoussystem development // cell differentiation

As shown in FIG. 6, gene expression amount of gene X-linked 1(NM_(—)018476) showed a linear decrease in PA, PD, and Parkin. FIG. 6 isa pattern graph and Heat map showing a result of Hierarchical Clusteringof the genes separated with K-mean Clustering Analysis.

The genes of Cluster 3 showed a linear increase of gene expressionamount in the order of PA<PD<Parkin, as shown in Table 8 and FIG. 7.

TABLE 8 Gene name Function Genbank accession # major histocompatibilitycomplex, antigen processing and presentation of NM_033554 class II, DPalpha 1 peptide or polysaccharide antigen via MHC class II // immuneresponse MHC class I polypeptide-related antigen processing andpresentation of NM_000247 sequence A peptide antigen via MHC class I //response to stress // immune response // cellular defense response //cell recognition // antigen processing and presentation pancreaticlipase-related protein 3 lipid catabolic process NM_001011709 secretedfrizzled-related protein 4 signal transduction // embryo implantationNM_003014 // Wnt receptor signaling pathway // cell differentiationSestrin 3 cell cycle arrest NM_144665 EGF-like repeats and discoidinI-like cell adhesion // multicellular organismal NM_005711 domains 3development //angiogenesis aldo-keto reductase family 1, memberprostaglandin metabolic process NM_003739 C3 (3-alpha hydroxysteroiddehydrogenase, type II)

FIG. 7 is a result of clustering of the genes which showed a linearincrease of gene expression in non-PD, PD, and Parkin.

2.4. Reprogramming of Functional Group of Human Biomarker CandidateAmong Non-PD vs. PD and Non-PD vs. Parkin

The functional groups among non-PD vs. PD and non-PD vs. Parkin werereprogrammed, and the human biomarker candidates were classified byusing the genes obtained from Gene Ontology and Panther database system(http://david.abcc.ncifcrf.gov/home.jsp) (FIGS. 8 a to 8 d). FIGS. 8 ato 8 d represented the number of genes which were reclassified asbiologically-functional groups from the genes having the gene expressionlevel of at least two-fold between the three groups of non-PD, PD,Parkin.

The biological categories included transcription factor, nucleic acidbinding, receptor, kinase, oxido-reduction protein, signal molecule,cell adhesion molecule and the like.

The genetic functional groups which showed up-regulation (FIG. 8 a) ordown-regulation (FIG. 8 b) in Idiopathic PD patient were compared withthose of non-PD patient (control group). The genetic functional groupswhich showed up-regulation (FIG. 8 c) or down-regulation (FIG. 8 d) inParkin (parkin deficiency) patient were compared with those of non-PDpatient (control group). These graphs represented human biomarkercandidate which were re-classified according to the biological functionsand notable up-regulation or down-regulation in idiopathic PD patientand parkin-deficiency PD patient.

2.5. Genes Regulated Differentially in Non-PD, PD and Parkin PatientsDue to the Oxidative Stress

It has not been known which genes show selective sensitivity to theoxidative stress and how those genes affect the cell. PD-related geneswhich are differentially regulated by the oxidative stress are analyzedin non-PD, PD and Parkin groups. The genes regulated differentially bythe oxidative stress were classified again as a functional group fromthe genes showing the gene expression level of at least two-fold betweenthe groups of non-PD, PD, and Parkin, and then the PD-related genes wereselected (refer to http://www.ncbi.nlm.nih.gov/pubmed/).

These groups included oxidoreductase, endoplasmicreticulum/ubiquitin-like, exocytosis/membrane trafficking,apoptosis/cell survival, structure/transport, translation,nuclear/transcriptional, and cell cycle. The PD-related genes in K-meanclustering 2 and 6 were classified again to change the cluster number.The genes which were differentially expressed between non-PD, PD andParkin because of the oxidative stress are summarized in Table 9.

TABLE 9 K-mean Putative Function clustering Group I: oxidoreductasealdo-keto reductase family 1, member B1 (aldose reductase) 6 (AKR1B1)Group II: endoplasmic reticulum/ubiquitin-like dopamine receptor D1(DRD1) 6 Group III: exocytosis/membrane trafficking majorhistocompatibility complex, class II, DP α1 3 (HLA-DPA1) synaptotagminXIV (SYT14) 6 Group IV: apoptosis/cell survival actin, α, cardiac muscle1 (ACTC1) 2 clusterin (CLU) 2 transforming growth factor, β3 (TGFB3) 6Group V: structure/transport major histocompatibility complex, class II,DP α1 3 (HLA-DPA1) transforming growth factor, β3 (TGFB3) 6 solutecarrier family 6, member 6 (SLC6A6) 6 aldo-keto reductase family 1,member B1 6 (aldose reductase) (AKR1B1) signal peptide, CUB domain,EGF-like 3 (SCUBE3) 6 Group VI: translation angiotensin II receptor,type 1 (AGTR1) 2 reelin (RELN) 6 G protein α14 (GNA14) 6 solute carrierfamily 6, member 6 (SLC6A6) 6 Group VII: nuclear/transcriptionalintegrin α2 (ITGA2) 6 transforming growth factor, β3 (TGFB3) 6 dopaminereceptor D1 (DRD1) 6 Group VIII: cell cycle heat shock 70 kDa protein 2(HSPA2) 6

Surprisingly, the selective gene expression of the groups in Clusters 2,3, and 6 increased linearly between non-PD, PD and Parkin patients, andthe groups belonged to AKR1B1 (oxidoreductase), DRD1 (endoplasmicreticulum/ubiquitin-like), HLA-DPA1 and SYT14 (exocytosis/membranetrafficking), ACTC1, CLU and TGFB3 (apoptosis/cell survival), HLA-DPA1,TGFB3, SLC6A6, AKR1B1 and SCUBE (structure/transport), AGTR1, RELN,GNA14 and SLC6A6 (translation), ITGA2, TGFB3 and DRD1(nuclear/transcriptional), and HSPA2 (cell cycle). The genbank accessionnumbers of the genes are summarized in the following table.

Gene name Gene accession number AKR1B1 NM_001628 DRD1 NM_000794 HLA-DPA1NM_033554 SYT14 NM_153262 ACTC1 NM_005159 CLU NM_001831 TGFB3 NM_003239SLC6A6 NM_003043 SCUBE3 NM_152753 AGTR1 NM_000685 RELN NM_005045 GNA14NM_004297 ITGA2 NM_002203 HSPA2 NM_021979

The obtained data can assist the understanding of mitochondrialdysfunction and oxidative stress in Idiopathic and Parkin-derivedParkinson's disease, and provide a useful guidance for investigation ofadditional functional properties.

2.6. Immortalization of Mesenchymal Stromal Cells Derived from AdiposeTissue of Parkinson's Disease Patient with pGRN145 Including hTERT

The cells of non-PD, PD, Parkin in Example 1.1 were spread again on the24-well plate to reach 90 percent of confluence without addingantibiotics on one day before transfecting. 50 μL of serum-free OPTI-MEMI Medium (Gibco BRL, Gaithersburg, Md.) including 1 μg of pGRN145 DNA(Geron Corporation, Menlo Park, Calif., USA), and 50 μL of OPT1-MEM IMedium including 2 mL of LIPOFECTAMINE LTX Reagent (Gibco) were mixedand added to each well, and replaced with new media after culturing at37° C. for 24 hr. After 48 hours, the transfected cells were cultured inmedia including Hygromycin-B (30 μg/mL) for 2 to 3 weeks, and the finalconcentration was reduced to be 10 μg/mL. The clones derived from onecell were selected.

The cell shapes of non-PD, PD, and Parkin cells belonging to theselected clones were compared before immortalization, afterimmortalization, 6-month culture and one-year culture with humantelomerase reverse transcriptase (hTERT). The result is described inFIG. 9. In FIG. 9, non-PD (PA) represents the shape of cell havingAccession No. KCLRF-BP-00239(PA1) before and after immortalization, PDis for the shape of cell having Accession No. KCLRF-BP-00241(PD1) beforeand after immortalization, and Parkin is for the shape of cell havingAccession No. KCLRF-BP-00243(Pakin 1) before and after immortalization.

The chromosomal structure of the cells obtained shortly after theimmortalization and after culturing the immortalized non-PD, PD, andParkin for a year were analyzed and shown in FIG. 10. The chromosomalstructure of the cells before immortalization was normal, and thus wasnot analyzed. From top to bottom in FIG. 10, non-PD (PA) represents thestate of immortalized cell having Accession Nos. KCLRF-BP-00239(PA1) andKCLRF-BP-00240(PA2), PD represents the state of immortalized cell havingAccession Nos. KCLRF-BP-00241(PD1) and KCLRF-BP-00242(PD2), and Parkinrepresent the state of immortalized cell having Accession Nos.KCLRF-BP-00243(Pakin1) and KCLRF-BP-00244(Pakin2).

Specifically, the cell division at metaphase of mitosis was restrainedwith colcemid (Gibco) Stoc solution. That is, the cells were collectedfrom the supernatant obtained by centrifuging at 1500 rpm, shocked with0.075M KCl hypotonic, and fixed with the addition of Canoy's fixativeincluding methanol and acetic acid at a mixing ratio of 3:1, and Giemsastaining GTG banding). The prepared cell slide was analyzed withKaryotype Analysis program: ChIPS-Karyo (Chromosome Image ProcessingSystem) (GenDix, Inc. Seoul, Korea), and the analyzed result is shown inFIG. 10.

As shown in FIG. 10, the immortalized cell showed abnormal nuclear typecompared with the non-immortalized cell.

2.7. Separation of Mitochondria from the Cultured Cell for MitochondriaComplex I, II, IV and Citrate Synthase Assays

The non-PD, PD, and Parkin cells immortalized with hTERT were washedwith PBS and suspended in 10 mM Tris, pH 7.6 including proteaseinhibitor cocktail. The cells were blocked with 1-mL syringe, added with1.5M sucrose and centrifuged at 600×g, 2° C. for 10 minutes. Then, thesupernatant were centrifuged again at 14,000×g, 2° C. for 10 minutes andthe obtained pellet were washed with protease inhibitor cocktail in 10mM Tris (pH 7.6). The mitochondria pellets were re-suspended in 10 mMTris (pH 7.6) including protease inhibitor cocktail and subsequentlypreserved in ice before use.

Complex I assay: The activity of complex I was analyzed withspectrometer at 600 nm by using 240 μL reagent including 25 mM potassiumphosphate, 3.5 g/L BSA, 60 μM DCIP, 70 μM decylubiquinone, 1.0 μMantimycine-A, and 3.2 mM NADH, pH 7.8.

Namely, the obtained mitochondria sample (1 μg/10 μL) was added to abuffer solution without NADH, incubated at 37° C. for 3 minutes, andthen added with 5 μL of 160 mM NADH. The absorbance was measured at 37°C. for 5 minutes at 30 second-intervals, and after 5 minutes, and 2.5 μLrotenone (100 μM of rotenone dissolved in 1 mM in dimethylsulfoxide and10 mM Tris, pH 7.6) was added thereto. Then, the absorbance was measuredat 37° C. for 5 minutes at 30 second-intervals. The results are shown inFIG. 11.

Complex II assay: The activity of complex II was analyzed withspectrometer at 600 nm with 240 μL reagent including 80 mM potassiumphosphate, 1 g/L BSA, 2 mM EDTA, 0.2 mM ATP, 10 mM succinate, 0.3 mMpotassium cyanide, 60 μM DCIP, 50 μM decylubiquinone, 1 μM antimycine-A,and 3 μM rotenone, pH 7.8.

Specifically, the obtained mitochondria sample (1 μg/10 μL) was added toa buffer solution without succinate and potassium cyanide, incubated at37° C. for 10 minutes, and then 204, of 1.5M succinate and 0.75 μL of0.1M KCN were added thereto. The absorbance was measured at 37° C. for 5minutes at 30 second-intervals, and BLANK was detected in the presenceof 5 mM malonate. The result is shown in FIG. 11.

Complex IV assay: The activity of complex IV was analyzed withspectrometer at 550 nm with 240 μL reagent including 30 mM potassiumphosphate, 2.5 mM dodecylmaltoside, and 34 μM ferrocytochrome c, pH 7.4

Specifically, the obtained mitochondria sample (1 μg/10 μL) was added toa buffer solution and shortly after, the absorbance was measured at 30°C. for 5 minutes at 30 second-intervals, and BLANK was detected in thepresence of 1 mM KCN. The result is shown in FIG. 11.

Citrate synthase assay: The activity of citrate synthase was analyzedwith spectrometer at 412 nm with 240 μL reagent (pH 7.5) including 50 mMTris-HCl, 0.2 mM 5,5′-dithiobis-(2-nitrobenzoic acid), 0.1 mM acetyl-CoAand 0.5 mM oxaloacetate.

Specifically, the obtained mitochondria sample (1 μg/10 μL) was added toa buffer solution without oxaloacetate, and incubated at 30° C. for 5minutes. After adding by 2.5 μL of 50 mM oxaloacetate, the absorbancewas measured at 37° C. for 5 minutes at 30 second-intervals. The resultis shown in FIG. 11. This assay began with the addition of oxaloacetate,and for a control group, water was added in the equal amount.

As represented in FIG. 11, the result of biochemical analysis formitochondrial respiration chain of immortalized cell confirmed that theactivities of PD and Parkin decreased compared with Non-PD activity.

2.8. Electron Microscopy Analysis

The non-PD, PD, and Parkin cells immortalized with hTERT were washedwith PBS and fixed with 0.1% glutaraldehyde and 4% paraformaldehyde inPBS at 4° C. for 2 hours. The cells were collected and centrifuged at2000×g, at 4° C. and for 3 minutes to obtain pellets. The preparedpellets were re-suspended in warm 1% agar and centrifuged at 2000×g at4° C. for 3 minutes to obtain the pellets again. Then, the pellets werewashed with PBS three times, and the cell pellets embedded with agarwere fixed again with 1% osmium tetroxide for 2 hours and washed withPBS three times. The cell pellets embedded with agar was dehydrated inethanol and fixed again with Epon 812. The ultrathin (70 nm) sectionswere collected on Formvar/carbon-coated nickel grids, stained with 2.5%uranyl acetate for 7 minutes and with lead citrate for 2.5 minutes, andthen were observed with JEOL JEM-1011 electron microscope. The resultsare shown in FIG. 12. The comparison of mitochondrial shape ofprimary-cultured non-PD, PD and Parkin cell and immortalized non-PD, PDand Parkin cell showed that the mitochondria shape of non-PD(non-Parkinson's disease) patient was normal, but those of PD(Idiopathic Parkinson's disease) patient and Parkin (parkin-deficientParkinson's disease) patient were damaged gradually. This suggested themitochondrial damage is an important cause of Parkinson's disease.

2.9. Western Blot Analysis

The cells (The primary-cultured mesenchymal stromal cells derived fromnon-PD, PD, and Parkin patients were used in FIG. 13 a, and immortalizedmesenchymal stromal cells derived from non-PD, PD, and Parkin patientswere used in FIGS. 13 b, 13 c, 14 a, 15 a, and 16 a) were washed withcold PBS and divided into lysis buffer (cell signaling) and PMSF. Thedivided products were centrifuged at 15,000×g at 4° C. for 20 minutes.The products were analyzed quantitatively with Bradford reagent(Bio-Rad, Hercules, Calif.). The protein which was used in the sameamount as other primary-cultured cells, and immortalized cells wereloaded on SDS-PAGE, transferred to PVDF membrane (Millipore), andblocked with 5% non-fat dry milk in TBST. The proteins on the membranewere detected by chemical luminescence with X-ray film using ECL-Plussubstrate (GE Healthcare, Buckinghamshire, USA). Antibodies of Hsp25,Hsp60 and Hsp90 were obtained from Santa Cruz Biotechnology (Santa Cruz,Calif., USA) and antibodies of DJ-1, P-mTOR, mTOR, P-S6K, S6K, LC3-I,and LC3-II were obtained from Chemicon (Temecula, Calif., USA).Prohibitin and β-actin (Santa Cruz) were used as internal control. Thewestern blot analysis was performed with National Institutes of Healthimage processing and analyzing program (ImageJ, v1.38;http://rsb.info.nih.gov/ij/).

The obtained results are shown in FIGS. 13 a-13 c, 14 a-14 c, 15 a-15 band 16 a-16 b. FIG. 13 a represents the change in the gene expression ofmitochondrial markers, DJ-1, Hsp60, and Hsp25 in the primary-culturedmesenchymal stromal cells derived from non-PD, PD, and Parkin patients.FIG. 13 b represents the change in the gene expression of mitochondrialmarkers, Hsp60 in immortalized mesenchymal stromal cells derived fromnon-PD, PD, and Parkin patients. FIG. 13 c represents the change in thegene expression of mitochondrial markers, Hsp90 in immortalizedmesenchymal stromal cells derived from non-PD, PD, and Parkin patients.FIG. 14 a represents the change in the gene expression of autophagymarker, mTOR, and S6K in immortalized mesenchymal stromal cells derivedfrom non-PD, PD, and Parkin patients. FIG. 14 b and FIG. 14 c are thegraphs showing the quantitative analysis of FIG. 14 a. FIG. 15 arepresents the change in the gene expression of autophagy marker, mTOR,and S6K in immortalized mesenchymal stromal cells derived from non-PD,PD, and Parkin patients. FIG. 15 b is the graph showing the quantitativeanalysis of FIG. 15 a. FIG. 16 a represents the change in the geneexpression of autophagy marker, LC3 (LC3-I, LC3-II) in immortalizedmesenchymal stromal cells derived from non-PD, PD, and Parkin patients.FIG. 16 b is the graph showing the quantitative analysis of FIG. 16 a.

1. A composition for diagnosing a disease, comprising a mesenchymalstromal cell derived from adipose tissue, wherein the disease isselected from the group consisting of Parkinson's disease, Alzheimer'sdisease, Huntington's disease, hereditary dystonia, hereditarydyskinesia, and metabolic disease.
 2. The composition according to claim1, wherein the composition is used for diagnosing the Parkinson'sdisease.
 3. A method of providing information for diagnosing a disease,comprising the steps of separating a mesenchymal stromal cell derivedfrom adipose tissue of a subject; assaying a gene expression pattern ofthe mesenchymal stromal cell; and determining the presence of a diseaseby analyzing the assayed result, wherein the disease is selected fromthe group consisting of Parkinson's disease, Alzheimer's disease,Huntington's disease, hereditary dystonia, hereditary dyskinesia, andmetabolic disease.
 4. The method of providing information for diagnosinga disease according to claim 3, wherein the disease is Parkinson'sdisease.
 5. The method of providing information for diagnosing a diseaseaccording to claim 4, wherein the disease of a subject is diagnosed tobe Parkinson's disease, in case there is an increased expression of atleast one gene in the mesenchymal stromal cells derived from the adiposetissue selected from the group consisting of ITGA8(Genbank accession No:NM_(—)003638), CTSH(Genbank accession No: NM_(—)004390), CCRL1(Genbankaccession No: NM_(—)178445), TGFB3(Genbank accession No: NM_(—)003239),DRD1(Genbank accession No: NM_(—)000794), GNA14(Genbank accession No:NM_(—)004297), PENK(Genbank accession No: NM_(—)006211), PRG4(Genbankaccession No: NM_(—)005807), LGR5(Genbank accession No: NM_(—)003667),HLA-DPA1(Genbank accession No: NM_(—)033554), SCUBE3(Genbank accessionNo: NM_(—)152753), HSPA2(Genbank accession No: NM_(—)021979),RELN(Genbank accession No: NM_(—)005045), EDNRB(Genbank accession No:NM_(—)000115), ITGA2(Genbank accession No: NM_(—)002203), SLC6A6(Genbankaccession No: NM_(—)003043), F2RL2(Genbank accession No: NM_(—)004101),CDK6(Genbank accession No: NM_(—)001259), AKR1B1(Genbank accession No:NM_(—)001628), MMP8(Genbank accession No: NM_(—)002424), ID1(Genbankaccession No: NM_(—)181353), NEFM(Genbank accession No: NM_(—)005382),ATP1B1(Genbank accession No: NM_(—)001677), TNFRSF11B(Genbank accessionNo: NM_(—)002546), and TNFRSF10D(Genbank accession No: NM_(—)003840), ora decreased expression of at least gene selected from the groupconsisting of BEX1(Genbank accession No: NM_(—)018476), IL8(Genbankaccession No: NM_(—)000584), and CXCL6(Genbank accession No:NM_(—)002993).
 6. A method of screening a drug treating Parkinson'sdisease, comprising the steps of: contacting a candidate compound with amesenchymal stromal cell derived from adipose tissue; and assaying agene expression pattern in the mesenchymal stromal cell, wherein thecandidate compound is determined as a drug treating Parkinson's disease,in case difference of gene expression pattern is observed in themesenchymal stromal cells between the treatment and non-treatment of thedrug candidate, and the gene expression pattern is obtained from one ormore selected from the group consisting of ITGA8(Genbank accession No:NM_(—)003638), CTSH(Genbank accession No: NM_(—)004390), CCRL1(Genbankaccession No: NM_(—)178445), TGFB3(Genbank accession No: NM_(—)003239),DRD1(Genbank accession No: NM_(—)000794), GNA14(Genbank accession No:NM_(—)004297), PENK(Genbank accession No: NM_(—)006211), PRG4(Genbankaccession No: NM_(—)005807), LGR5(Genbank accession No: NM_(—)003667),HLA-DPA1(Genbank accession No: NM_(—)033554), SCUBE3(Genbank accessionNo: NM_(—)152753), HSPA2(Genbank accession No: NM_(—)021979),RELN(Genbank accession No: NM_(—)005045), EDNRB(Genbank accession No:NM_(—)000115), ITGA2(Genbank accession No: NM_(—)002203), SLC6A6(Genbankaccession No: NM_(—)003043), F2RL2(Genbank accession No: NM_(—)004101),CDK6(Genbank accession No: NM_(—)001259), AKR1B1(Genbank accession No:NM_(—)001628), MMP8(Genbank accession No: NM_(—)002424), ID1(Genbankaccession No: NM_(—)181353), NEFM(Genbank accession No: NM_(—)005382),ATP1B1(Genbank accession No: NM_(—)001677), TNFRSF11B(Genbank accessionNo: NM_(—)002546), TNFRSF10D(Genbank accession No: NM_(—)003840),BEX1(Genbank accession No: NM_(—)018476), IL8(Genbank accession No:NM_(—)000584), and CXCL6(Genbank accession No: NM_(—)002993).
 7. Themethod of screening a drug treating Parkinson's disease according toclaim 6, wherein the drug candidate is determined as a drug treatingParkinson's disease, in case the treatment of drug candidate results inan increased expression of at least one gene selected from the groupconsisting of ITGA8(Genbank accession No: NM_(—)003638), CTSH(Genbankaccession No: NM_(—)004390), CCRL1(Genbank accession No: NM_(—)178445),TGFB3(Genbank accession No: NM_(—)003239), DRD1(Genbank accession No:NM_(—)000794), GNA14(Genbank accession No: NM_(—)004297), PENK(Genbankaccession No: NM_(—)006211), PRG4(Genbank accession No: NM_(—)005807),LGR5(Genbank accession No: NM_(—)003667), HLA-DPA1(Genbank accession No:NM_(—)033554), SCUBE3(Genbank accession No: NM_(—)152753), HSPA2(Genbankaccession No: NM_(—)021979), RELN(Genbank accession No: NM_(—)005045),EDNRB(Genbank accession No: NM_(—)000115), ITGA2(Genbank accession No:NM_(—)002203), SLC6A6(Genbank accession No: NM_(—)003043), F2RL2(Genbankaccession No: NM_(—)004101), CDK6(Genbank accession No: NM_(—)001259),AKR1B1(Genbank accession No: NM_(—)001628), MMP8(Genbank accession No:NM_(—)002424), ID1(Genbank accession No: NM_(—)181353), NEFM(Genbankaccession No: NM_(—)005382), ATP1B1(Genbank accession No: NM_(—)001677),TNFRSF11B(Genbank accession No: NM_(—)002546), and TNFRSF10D(Genbankaccession No: NM_(—)003840), or a decreased expression of at least onegene selected from the group consisting of BEX1(Genbank accession No:NM_(—)018476), IL8(Genbank accession No: NM_(—)000584), andCXCL6(Genbank accession No: NM_(—)002993), relative to non-treatment ofthe drug candidate.